Participants were required to report to the laboratory on four separate visits. During the first visit, anthropometric measurements were taken along with the estimation of daily total energy expenditure and the assessment of peak exercise responses. The second, third, and fourth visits served as experimental conditions. The experimental conditions used an experimental design with a repeated-measures statistical plan, in which each participant performed all three experimental conditions: Low-intensity (LI), Low-intensity with BFR (LI-BFR), and High-intensity (HI). Each experimental condition consisted of three phases: pre-exercise resting phase, exercise phase, and recovery phase. All conditions were the same except for the exercise protocol.

Participants were informed of the risks and benefits of the study, prior to participation, and written informed consent was obtained from all individuals participating in the study. The study complied with the standards set by the Declaration of Helsinki and was approved by the University's Institutional Review Board (IRB #: 2020.38). All participants recruited for this study had no diagnosed cardiovascular, pulmonary, or metabolic disease, and were not taking medications that influenced cardiovascular responses, based on their responses to a health history questionnaire. Participants were excluded if they self-reported a history of metabolic, pulmonary, cardiovascular disease, or an orthopedic-related injury in the past 6 months. In addition, participants were asked to refrain from any unaccustomed strenuous physical activity, maintain their normal dietary habits, and not to take any anti-inflammatory drugs or nutritional supplements during the experimental period.

There were 11 participants recruited for this study; however, ten of the participants were included in the analysis. One participant's data was excluded due to an equipment error during one of their visits. Power calculations were performed using effect sizes from a previous study that examined EPOC and fat oxidation with interval exercise (26). The most conservative effect size (d = 0.6) indicated that a sample size of 7 subjects would be sufficient what α = 0.05 and β = 0.80. The sample size recruited in this study is comparable to other studies examining acute protocols with a crossover design (26, 27). Among the ten participants in the study, there were seven males and three females. Participant characteristics are shown in Table 1. The total work output was 2,421.6 ± 567.6 W in each exercise protocol. The equal total work outputs were part of the design of the study.

The experimental conditions were performed on different days separated by at least 48 h and assigned in a randomized order (http://www.randomization.com). Each participant performed the experimental conditions at approximately the same time in the morning. Environmental conditions of the experimental visits are shown in Table 2. Three hours prior to each experimental condition, participants were asked to consume a standardized meal, based on an estimation of their total energy expenditure. For the purposes of the standardized meal, total estimated energy expenditure consisted of estimations of resting energy expenditure via the Nelson equation (31, 32) and estimation of daily physical activity energy expenditure, as determined by responses to the International Physical Activity Questionnaire (IPAQ) (33). The total caloric intake of the standardized meals was approximately 15%–20% of the participant's total estimated energy expenditure. This percentage is equivalent to the percentage of calories consumed by an average adult during breakfast (34). The standardized meal consisted of a nutritional shake (Ensure® Original Nutrition Shake, 220 calories per serving, 33 g carbohydrates, 6 g fat, and 9 g protein) and a cereal bar (Kroger® Fruit & Grain Cereal Bar, 130 calories per serving, 25 g of carbohydrates, 3 g of fat, and 1 g of protein). The number of servings of the nutritional shake and cereal bars were adjusted to ensure each participant consumed between 15% and 20% of their estimated total energy expenditure. The mean calories consumed in the standardized meal was 434.0 ± 68.0 kcals (76.3 ± 12.2 g carbohydrates, 11.4 ± 1.8 g of fat, 11.5 ± 2.5 g of protein). In addition to consuming the standardized meal, participants were asked to avoid consumption of caffeine, alcohol, and tobacco and avoid vigorous activity for at least 12 h and 24 h prior to each experimental condition, respectively. Adherence to all pre-condition requirements, including consuming the entire assigned meal, were verbally confirmed by the investigator prior to the start of the experimental conditions.

In order to control for daily variations in resting VO₂, the participants were asked to rest in a semi-recumbent position for 30 min for each experimental condition. After the first 15 min of the resting period, the participants were fitted with a facemask, and breath-by-breath gas exchange and heart rate (HR), via a chest strap HR monitor, were continuously collected for the final 15 min of the resting period.

The exercise protocols were matched for total work output and were performed on an electronically-braked cycle ergometer (Corival, Lode, Netherlands). Participants were instructed to maintain a pedal rate of 60–80 RPM throughout the protocol. Each exercise protocol started with a four-minute warm-up (20W). For the LI-BFR and LI exercise conditions, participants completed 10 two-minute work intervals interspersed with one-minute recovery. Work intervals were performed at a cycling work rate that elicited a VO₂ corresponding to 70% of VT, as determined from the GXT. The workload for the recovery intervals was 20 W. During the LI-BFR condition, the BFR cuffs (SC10D, Hokanson, Bellevue, WA, 10.0 cm width) were placed proximally on both legs and inflated to 80% of limb occlusion pressure (LOP) at the start of the work intervals and remained inflated throughout each work interval. While the degree of restriction has been shown to effect cardiovascular responses (35), at limb occlusion pressures above 60%, muscle responses become pressure-dependent, with local muscle oxygenation decreasing and neuromuscular activation and blood lactate increasing during low-intensity exercise (36–38)., Therefore, 80% LOP was used throughout each occlusion condition, which is in line with previous research (15). During all recovery intervals, the cuffs were rapidly deflated and remained deflated until the next work interval. For the HI, participants completed five two-minute work intervals interspersed with one-minute recovery. Work intervals were performed at a work rate corresponding to 140% of VT, as determined from the GXT. This work:rest ratio was utilized so that the total work performed during all conditions was equal. During all recovery intervals, the power output was reduced to 20 W. Breath-by-breath gas exchange and HR, via a chest strap HR monitor, were continuously collected throughout each exercise protocol. Baseline VO₂ was considered the average VO₂ over the final 10 min of rest.

Immediately following each exercise protocol, participants assumed a semi-recumbent position and were asked to remain seated for the 3-hour recovery period in order to measure EPOC and substrate utilization. Immediately at the start of recovery, breath-by-breath gas exchange was collected continuously for 30 min, except for a short break at 15 min for the participant to drink water. Following the first 30 min, gas exchange was collected for 8 min of every 15-min period between 30 and 180 min. While the gas exchange was not being collected the participants could drink water ad-libitum while remaining in the semi-recumbent position. HR was continuously collected over the entire recovery period. Participants were allowed two breaks from the semi-recumbent position at 60 min and 120 min to use the bathroom, if necessary.

To determine the BFR pressure for each participant, cuffs (SC10D, Hokanson, Bellevue, WA, 10.0 cm width) were placed around the proximal portion of both thighs. Participants laid supine on a treatment table and rested for five minutes. Then the posterior tibial artery was identified on the participant's dominant leg using Doppler auscultation (Nicolet Imex Pocket Dop II, Natus, San Carlos, CA). Once the pulse was identified, the cuffs were rapidly inflated to 50mmHg and then progressively inflated until the pulse was eliminated. The pressure associated with the cessation of the pulse was taken as the limb occlusion pressure (LOP) (6). This procedure was performed prior to all exercise protocols that involved the application of BFR. During all protocols that involved BFR, the cuffs were inflated to each participants' custom pressure based on their LOP (80% LOP) (15).

The magnitude of EPOC was calculated over the first 90 min of recovery. For each condition, the absolute VO₂ (L·min⁻¹) was averaged over the final 10 min of the resting period and was considered the baseline measurement for that condition. During recovery, VO₂ was averaged over 15-s during the following times: 0–15 min, 17–30 min, 38–45 min, 53–60 min, 68–75 min, and 83–90 min. For each 15-s VO₂, a netVO₂ was calculated by subtracting the baseline VO₂ from the 15-s VO₂. The netVO₂ was then plotted against the time during recovery and EPOC was calculated as the area under the curve via the trapezoidal rule.

During each condition breath-by-breath VO₂ (L·min⁻¹) averaged every 15 s from the start of the first work interval to the end of the last work interval. For each 15-s VO₂, a netVO₂ was calculated by subtracting the baseline VO₂ from the 15-s VO₂. Exercise excess energy expenditure and recovery excess energy expenditure were calculated from the netVO₂ (L·min⁻¹) during exercise and the first 90 min of recovery, respectively. Due to limitations of using RER-based caloric equivalents during high-intensity exercise, net VO₂ (L·min⁻¹) was multiplied by a constant factor of 5 kcal·L⁻¹ O₂ for all conditions (pre-exercise, exercise, and recovery) to estimate energy expenditure. The exercise and recovery excess energy expenditures were summed to calculate the total excess energy expenditure.

Fat oxidation rates and carbohydrate oxidation rates were calculated from the average VO₂ and VCO₂ from the final 10 min of rest and 10-minute averages during recovery between 50 and 60 min (60 MIN), 110–120 min (120 MIN), and 170–180 min (180 MIN). Calculating fat oxidation and carbohydrate oxidation by indirect calorimetry assumes steady-state conditions which may not be present during the first 60 min of recovery from high-intensity exercise. Bicarbonate buffering and non-metabolic CO₂ have been shown to be no different from resting control conditions from 60 to 120 min after high-intensity exercise (39). Therefore, exercise and data early in recovery (first 50 min of recovery) were excluded from the analysis. Fat and carbohydrate oxidation rates were calculated based on equations developed by Peronnet and Massicotte (40). The equations were as follows:Fatoxidationrate(g⋅min-1)=1.695⋅VO2–1.701⋅VCO2Carbohydrateoxidationrate(g⋅min-1)=4.585⋅VCO2–3.226⋅VO2

Statistical analyses were completed using IBM SPSS statistical software (Version 25.0; SPSS, Inc., Chicago, IL). Shapiro–Wilk test was conducted prior to all ANOVA analyses to assess the normality of the data. The test was non-significant in all cases, indicating no violations of normality for any variable. Additionally, test-retest reliability during rest was assessed using the intraclass correlation coefficient (ICC). A two-way mixed-effects model with absolute agreement was used, and ICC was calculated for average measures, reflecting the reliability of the mean measurement across conditions. ICC values were interpreted as: <0.50 = poor, 0.50–0.75 = moderate, 0.75–0.90 = good, and >0.90 = excellent reliability.

A two-way (condition [LI-BFR, HI, LI] by time [Rest, Ex, 60 MIN, 120 MIN, 180 MIN) repeated measures ANOVA was used to compare the absolute VO₂ (L·min⁻¹), relative VO₂ (mL·kg⁻¹·min⁻¹), RER, and HR. A two-way (condition [LI-BFR, HI, LI] by time [Rest, 60 MIN, 120 MIN, 180 MIN) repeated measures ANOVA was used to compare the fat oxidation rate and carbohydrate oxidation rates. A two-way (condition [LI-BFR, HI, LI] by time [exercise, recovery) repeated measures ANOVA was used to compare exercise and recovery excess energy expenditure between conditions. A one-way repeated measures ANOVA was used to compare total excess energy expenditure and EPOC between conditions. Subsequent Bonferroni pairwise post-hoc comparisons were made when necessary. Cohen's f and d were used as an estimate of effect size for the ANOVA and subsequent post-hoc comparisons, respectively. Cohen's f values were interpreted as follows: a small effect was defined as <0.25, a medium effect as 0.25–0.40, and a large effect as >0.40. Cohen's d values were interpreted as follows: a small effect was defined as <0.40, a medium effect as 0.40–0.75, a large effect as 0.75–1.10, a very large effect as 1.10–1.45, and a huge effect as >1.45 (41). Statistical significance was established if p ≤ 0.05.

The physiological responses (absolute VO₂ (L·min⁻¹), relative VO₂ (mL·kg⁻¹·min⁻¹), VCO₂ (L·min⁻¹), RER, and HR during each condition are shown in Table 3. Absolute VO₂ (L·min⁻¹), relative VO₂ (mL·kg⁻¹·min⁻¹), VCO₂ (L·min⁻¹), and RER did not meet the assumption of sphericity, so a Greenhouse-Gasser test was performed. There was a significant condition by time interaction for absolute VO₂ (L·min⁻¹) (p < 0.001; f = 2.64), relative VO₂ (mL·kg⁻¹·min⁻¹) (p < 0.001; f = 2.22;), VCO₂ (L·min⁻¹) (p < 0.001; f = 1.93), RER (p < 0.001; f = 1.38;), and HR (p < 0.001; f = 1.89). The decomposition of the interaction showed that while pre-exercise VO₂ (absolute or relative) did not differ between conditions (resting ICC = .900 and.912, respectively), during exercise, LIBFR was significantly higher compared to HI and LI (for all comparisons; 0.61 ≤ d ≤ 1.86). It then decreased after 60 min of recovery, only to become significantly higher again at the 120-minute recovery period compared to HI (d = 0.84 and d = 0.75, respectively) and LI (d = 1.07 and d = 0.87, respectively). Note that HI was also significantly higher compared to LI during exercise. The difference between HI and LI at 120 MIN were not significant. There were no differences between conditions for absolute VO₂ (L·min⁻¹) and relative VO₂ (mL·kg⁻¹·min⁻¹) at 60 MIN and 180 MIN.

Furthermore, VCO₂ (L·min⁻¹) at rest was not different between conditions (ICC = 0.879). During exercise, VCO₂ (L·min⁻¹) was higher in HIE than BFR (p = .003, d = 1.51) and LIE (p < .001, d = 2.12), and higher in BFR than LIE (p < .001, d = 2.27). At 120-min recovery, VCO₂ VCO₂ (L·min⁻¹) was higher in BFR than LIE (p = .029, d = 1.04), but similar to HIE (p = 0.083). The difference in VCO₂ (L·min⁻¹) at 120-recovery between HIE and LIE was not significant (p = 1.00). No other pairwise differences were significant at 60-min, or 180-min recovery.

The RER at rest was not different between conditions, but the RER during exercise was significantly different between all conditions (for all comparisons; 1.14 ≤ d ≤ 5.03). The only difference in RER between conditions during recovery occurred at 60 MIN. Specifically, the RER during LI-BFR was significantly lower than the RER during HI (d = 0.69) and LI (d = 0.40).

There were no differences between groups for HR at rest. There was a significantly greater HR during exercise in LI-BFR and HI compared to LI (d = 1.83 and d = 1.82, respectively); the difference between LI-BFR and HI was not significant. During recovery, HR at 120 MIN in LI- BFR was significantly greater than HI (d = 0.38); HR at 60 MIN and 180 MIN were similar between LI-BFR and HI. Additionally, HR during LI-BFR was significantly greater than LI at all points during recovery (60 MIN; d = 0.90; 120 MIN; d = 0.57; 180 MIN; d = 0.48). The only difference in HR during recovery between HI and LI occurred at 60 MIN (d = 0.95).

Descriptive values for ventilation (VE), expired fraction of oxygen (FeO₂), and expired fraction of carbon dioxide (FeCO₂) are presented in Table 4. These variables provide additional physiological context for the metabolic responses across conditions and time points. Because these measures were not part of the primary research purpose, no statistics were performed on VE, FeO₂, or FeCO₂, and they are reported descriptively only.

Fat oxidation rates at rest and during recovery for each condition are shown in Figure 1. There was a significant condition by time interaction for fat oxidation rate (p = 0.006; f = 0.62,). The decomposition of the interaction revealed that fat oxidation rate was similar at rest between conditions (ICC = 0.590); however, fat oxidation rate was significantly greater in LI-BFR compared to HI at 120 MIN (p = 0.002; d = 1.13). There were no other differences in fat oxidation rates between LI-BFR and HI; the difference at 60 MIN approached, but did not reach significance (p = 0.065; d = 0.92). Additionally, fat oxidation was significantly greater in LI-BFR compared to LI at 60 MIN (p = 0.015; d = 1.93) and 120 MIN (p = 0.002; d = 1.70;); the difference at 180 MIN was not significant. Fat oxidation rate was similar between HI and LI during all points in recovery. Within all conditions, fat oxidation rate during rest was significantly lower than all points during recovery (for all comparisons; p < 0.001; 3.56 ≤ d ≤ 5.70).

Carbohydrate oxidation rate responses at rest and during recovery for each condition are shown in Figure 2. The condition by time interaction and the main effect of condition were not significant (p = 0.623; resting ICC = 0.711). There was a significant main effect of time (p < 0.001; f = 2.42). Specifically, the carbohydrate oxidation rate was significantly greater during rest than at all points during recovery (p < 0.001).

The magnitude of EPOC in each condition is shown in Figure 3. The one-way ANOVA showed a significant main effect (p = 0.011; f = 0.85). The magnitude of EPOC in the LI-BFR condition was similar to HI; LI-BFR and HI produced greater EPOC compared to LI (d = 0.97 and d = 0.97, respectively).

Exercise, recovery, and total excess energy expenditure in LI-BFR, HI, and LI are shown in Figure 4. There was a significant condition by time interaction for exercise and recovery energy expenditure (p < 0.001; f = 3.32). One-way ANOVAs showed a significant effect for total excess energy expenditure (p < 0.001; f = 2.62). The decomposition of the interaction revealed a greater excess energy expenditure during exercise in LI-BFR compared to HI (p < 0.001; d = 3.58) and LI (p < 0.001; d = 1.41). Additionally, LI produced greater excess energy expenditure during exercise compared to HI (p < 0.001; d = 2.17). During recovery, there was no difference in excess energy expenditure between LI-BFR and HI, but both were different from LI ((p = 0.027; d = 1.68 and (p = 0.016; d = 1.68, respectively). One-way ANOVAs showed a significant effect for total excess energy expenditure (p < 0.001; f = 2.62). LI-BFR produced the greatest total excess energy expenditure (LI-BFR vs. HI: p < 0.001; d = 3.23; LI-BFR vs. LI: p < 0.001; d = 2.21). Total excess energy expenditure was also different between HI and LI (p = 0.027; d = 1.01).